Lipids are potentially useful as carriers for delivery of therapeutic molecules, particularly for delivery of nucleic acids. Lipids form liposomes, which can encapsulate, complex, or entrap nucleic acid molecules and thereby enhance delivery of this class of therapeutic molecules to target cells upon administration, e.g., intravenously to the circulation. Their usefulness in pharmaceutical compositions is limited by available methods to produce lipid-nucleic acid nanoparticles reproducibly. Various methods have been devised to produce such nanoparticles.
One method to produce nanoparticles consisting solely of lipids (vesicles) simply and reproducibly without sonication utilizes the ethanol injection described by Batzri et al., 1973, Biophys Biochem Acta 298:1015-19, and Kremer et al., 1977, Biochemistry 16:3932-35, whereby lipids solubilized in ethanol are injected into an aqueous solution to spontaneously form liposomes.
Van Buitenen et al. U.S. Pat. No. 7,468,151 describe a closed circuit system for sterilizing microparticles, including liposomes. The circuit system includes of a mixing chamber connected to a transflow filtration (TFF) unit. The TFF unit purifies a liquid dispersion of microparticles under aseptic conditions. The liquid is pumped aseptically from the mixing chamber through the TFF. The material retained in the TFF (the retentate) is recycled through the mixing chamber and the TFF unit until purified. The purification process is performed aseptically in one apparatus without removing the microparticles in the TFF retentate.
Others describe the process of producing nucleic acid-liposome particles by using specific methods to combine lipids and nucleic acids. Hirota et al., 1999, BioTechniques 27:286-89, teaches that liposomes coated with nucleic acids molecules spontaneously form when cationic lipids in ethanol are injected into an aqueous solution of nucleic acid. Maurer et al., 2001, Biophysical J, 80:2310-26 and Maurer et al. U.S. Pat. No. 7,094,423 teach a method of encapsulating nucleic acid molecules in a liposome. This method involves use of a preformed liposome comprising a cationic lipid. The liposome is destabilized by adding ethanol to the aqueous solution. Nucleic acid molecules are added to the destabilized lipid. Upon removal of ethanol, the liposome encapsulates the nucleic acid while reforming. An alternative method to encapsulate nucleic acids in liposomes is taught by Semple et al., 2001, Biophys Biochem Acta 1510:152-66 and Semple et al. U.S. Pat. No. 6,858,225. This method increases encapsulation efficiency by using an ionizable cationic lipid to form liposomes. An ethanol solution of lipids is combined with nucleic acids in an aqueous solution buffered at low pH. Ethanol is then removed while raising the pH to neutral value. Both methods require further processing of the resulting liposomes because aggregation during reconstitution produces a wide variation in size.
MacLachlan et al. U.S. Pat. No. 7,901,708 describes a process and an apparatus for producing uniform sized liposomes that encapsulate a nucleic acid. A stream RNA in an aqueous buffer is mixed with a stream of cationic lipids in ethanol at approximately equal flow rates in a T connector, in which lipid vesicles form instantly in a high ethanol concentration (45%). The solvent and solute concentrations are kept constant throughout the mixing process. No static mixers are involved. in which the liposomes are diluted. The stable nucleic acid liposomes are sterilized at the end of the process, immediately before a sterile fill step.
The methods described above require extensive labor to minimize bacterial contamination during the process of producing liposomes, including autoclaving, washing, and satisfying regulatory burdens. There remains an unmet need for a manufacturing method to encapsulate nucleic acids without the need for extensive mechanical processing steps to prepare preformed liposomes and without the need for processing step to reduce lipid-nucleic acid particles to a monodisperse population.